Electrospray ionization mass analysis apparatus and system thereof

ABSTRACT

Because of a low flow rate of the micro LC/MS, the dead volume or the diameter of a capillary tube must be minimized, and a sample and salt are likely to deposit in the capillary tube, with the result that clogging of the capillary tube and ESI nozzle often occurs. An electrospray ionization mass analysis apparatus and its system of the present invention predicts clogging, permit earlier cleaning or parts replacement, and detect clogging even if it has occurred, thereby suspending measurement and preventing samples from being introduced into an injector, with the result that waste of samples is avoided and effective data is ensured. 
     The aforementioned electrospray ionization mass analysis apparatus directly coupled to the micro LC prevents a micro LC, piping and ESI capillary tube from being clogged, and records an alarm in the data and stops the system whenever clogging has occurred, whereby highly reliable direct coupling with micro LC is ensured. 
     In the aforementioned electrospray ionization mass analysis apparatus and its system, a sample solution from a chromatograph is introduced into a capillary tube, and an electrospray ion source arranged for generating ions under atmospheric pressure generates ions, which are led into a mass spectrometer disposed in a vacuum chamber where the ion is subjected to mass analysis. The current value or strength of the ion having a specified mass in the sample solution is measured, and, when the current value has reduced below a threshold value, an error state is displayed.

FIELD OF THE INVENTION

The present invention relates to an electrospray ionization massanalysis apparatus and system thereof, wherein a sample solution elutingout of a low flow rate chromatograph such as a micro liquidchromatograph is led to an electrospray ion (ESI) source and is ionizedtherein, and the ions generated in this ion source are fed to a massspectrometer arranged in a highly vacuum space, where the ions aresubjected to mass analysis.

BACKGROUND OF THE INVENTION

In recent years there has been a remarkable growth in biologicalresearches over diversified fields. Especially, protein, peptide and DNAplay an extremely important role in the living body, and have been theobjects of study by a great number of research workers. Generally, theseorganic compounds derived from living organism occur in a very smallamount in complicated matrices. There has been a growing demand forextracting a very small amount of these biological organic compoundsfrom the living body and analyzing them using a mass spectrometerdirectly coupled with liquid chromatograph LC/MS apparatus) with a highdegree of sensitivity. The LC/MS apparatus is an apparatus forseparating a mixture with a liquid chromatograph (LC) and providingqualitative and quantitative analysis using a mass spectrometer (MS)with a high degree of sensitivity. Electrospray ionization (ESI) istypical ionization means used in the LC/MS. The ESI is ionsizationtechnique used under atmospheric pressure and is known as providing softand highly sensitive ionization. For this reason, this method has cometo be used very often for biological analysis.

To ensure stable and highly sensitive measurement of a very small amountof components using the aforementioned ESI, some parameters must beoptimized. One of these parameters is the flow rate that determines theamount of solution to be supplied to the ESI ion source. To achievehighly sensitive measurement, the flow rate of the solution flowingthrough the ESI capillary tube must be kept within a certain range. InESI, the optimum flow rate is said to lie in the range from 10 nL/min.(10⁻⁸ L/min) to several μL/min (10⁻⁶ L/min). If a solution is fed intothe ESI capillary tube at a flow rate higher or lower than this level,the ESI ionization will become unstable and anticipated highly sensitivemeasurement will not be achieved. U.S. Pat. No. 5,504,329 discloses anart for ESI improvement for providing highly sensitive measurement of avery small amount of components. The art disclosed therein was latercalled Nanospray technique. After the tip of an extra-fine capillarytube made of glass having an outer diameter of about 0.2 mm and innerdiameter of about 0.03 mm has been elongated by a burner or sharpened byetching, the nozzle tip is gold plated. The D.C. voltage of about 1 kVsupplied from the high voltage source is applied to the tip of thenozzle. The flow rate of a sample solution from a nanospray deviceranges from is several nL/min (several 10⁻⁹ L/min.) to 10 nL/min(several 10⁻⁸ L/min.). Measurement for more than one hour was enabled byonly the sample sucked into the nanospray spray capillary tube.Accordingly, this nanospray technique has come to be used in combinationwith extra-low flow rate chromatography in CE (CapillaryElectrophoresis); further, it has come to be used for extremely highlysensitive measurement of isolated components. The nanospray techniquehas enabled ESI measurement in the range of flow rate below 10 nL/min.

In the micro LC field, the flow rate is extremely small, below severalμL/min. and a big problem is raised by the dead volume of the LC partsand the pipe connection among the parts thereof. When the dead volumebetween the micro-column and detector is greater for the flow rate, thesample components separated by the micro-column will be dispersed andmixed among them, with the result that separation and sensitivity willbe lost a substantially. Further, the dead volume between the LC pumpand micro-column will cause a problem of the delay in gradient elution.This requires the dead volume to be minimized.

Gradient elution is a method for quick elution of the sample componentby changing the composition of the eluent with the lapse of time. Thisgradient elution technique is improves the separation of the samplecomponents. This improves the S/N ratio and reduces the measurement timeat the same time. Accordingly, LC is used extensively.

In micro LC, even if the start of gradient is specified and multiplepumps have fed out solvent at a predetermined flow rate, a long time isrequired before the composition of the eluent is changed in themicro-column. This delay raises a problem. This is called a delay ingradient elution.

Assume that a pump 1 is now feeding out solvent A at 20 μL/min. Alsoassume that a pump 2 starts to feed out solution B at the rate of 0.2μL/min. at a predetermined time. A mixer and a pipe regionrrangedbetween pumps 1 and 2 and micro-column. If their volume is 5 μL, thedelay of gradient will be 5/0.2=25 min. Namely, gradient is effectivelystarted in the micro-column 25 minutes after the pump 2 started to feedsolution B. This makes it difficult to ensure correct separation andanalysis by micro LC. In order to improve this delay of gradientelution, it is important to reduce the size of the mixer and deadvolume. The dead volume can be decreased by reducing the pipe diameteror pipe length. However, reduction of pipe diameter raises a new problemof easy clogging of the pipe. Especially when a biological sample is tobe analyzed, a biological macromolecule such as sugar and proteinpresent in the sample as well as NaCl and salts will cause clogging ofthe pipe. Further, separation of protein requires salt having a highconcentration of 100 mM or more to be added to the mobile phase in manycases. This salt of high concentration is deposited in the dead volumeof the pipe, with the result that the pipe is clogged in the finalstage. Accordingly, the frequently used system in the micro LC is amicro LC system where A semimicro or conventional LC pump is used up togradient solution feeding, and the eluent is split immediately beforethe inlet. A great volume (1 mL/min. to 0.1 mL/min.) of solvent is usedup to the pump, mixer and pipe, so the dead volume among them can beignored. In other words, the problem of delay in gradient elution hasbeen solved. The split eluent at a very small flow rate (10 to severalμL/min.) is led to the micro column through the injector. This methodhas a disadvantage that the greater part of solvent must be discarded bythe splitter, but it solves the aforementioned problem of the delay ingradient resulting from dead volume, and ensures economicalconfiguration of the system. For these merits, this method has come tobe used over a wide range.

The Japanese Application Patent Laid-Open No. 06-13015 discloses ionsimplantation apparatus for evaluating a trouble such as equipmentfailure, displacement by comparing with the reference value the statusvalue of a particular peak in a mass spectrum. The Japanese ApplicationPatent Laid-Open No. 10-10109 discloses an apparatus for avoiding damageof the optical detector cell resulting from a clogged flow path in amass analysis apparatus directly coupled with a liquid chromatograph,the aforementioned mass analysis apparatus being designed to ionize anddetect the component leaching therefrom.

The micro LC wherein the solvent is split before the micro column can besaid as an extension of the general-purpose LC and semimicro LCtechnology. So since the micro LC is capable of analyzing a tracequantity of sample, it is expected to find a widespread use in the fieldof biological technologies. According to this method, however, the majorportion of solvent is split and discarded as waste, and the amount ofsolvent flowing into the micro column is no more than one hundredth toone tenth of the solvent supplied to the splitter. So even if the ESIcapillary are clogged by salt or protein and the solvent cannot be ledto the micro column, solvent only flows to the waste liquid. Sincesolvent pressure is released to atmospheric pressure by the splitter,pressure is not changed by clogging of the micro column. Thus, cloggingof the analysis column or ESI capillary is not detected, with the resultthat the sample will be continuously fed from the automatic sampler

Since the clogging of the micro column or piping is not detected. Nosolvent flows in the vicinity of the injector, and washing is notcarried out by solvent, so the automatic sampler and injector will becontaminated by the sample. A large amount of data file from which anymass spectrum or chromatogram cannot be acquired will be stored in thememory of a control data processor. What is more crucial is thatprecious samples will be consumed in vain by clogging of the microcolumn. Further, it is not clear when the micro column was clogged, withthe result that data reliability will be placed under suspicion.

Further, the aforementioned Laid-open Publication does not disclose anymeans for detecting the clogging of a capillary tube or ESI nozzlecaused by deposition of salts due to low flow rate and for suspendingmeasurement, thereby avoiding waste of samples in the micro LC/MS, orany specific device for predicting the possible clogging of thecapillary tube.

DISCLOSURE OF THE INVENTION

The object of the present invention is to provide an electrosprayionization mass analysis apparatus and the system thereof that ensureeffective data at all times during measurement, by utilizing means fordetecting the clogging of a capillary tube or ESI nozzle caused bydeposition of salts due to low flow rate and for suspending measurement,thereby avoiding waste of samples in the micro LC/MS, or by predictingthe possible clogging of the capillary tube.

In an electrospray ionization mass analysis apparatus directly coupledwith a micro LC, the present invention prevents clogging of the micro LCcolumn, piping and ESI capillary, and records the alarm in the data andstops the system whenever any clogging has occurred, thereby ensuringhighly reliable direct connection with the micro LC.

The present invention provides an electrospray ionization mass analysisapparatus wherein;

eluate from a chromatograph is introduced into the capillary tube, and

an electrospray ion source arranged for generating ions underatmospheric pressure generates ions, which are led into a massspectrometer disposed in a vacuum chamber where the mass spectrum isgiven. This electrospray ionization mass analysis apparatus ischaracterized in that the current value of the ion of a specific mass ismonitored, and, when this ion current value has reduced below athreshold value, a flag is set to indicate an error.

Further, the present invention provides an electrospray ionization massanalysis apparatus wherein eluate from a chromatograph is introducedinto the capillary tube, and an electrospray ion source arranged forgenerating ions under atmospheric pressure generates ions, which are ledinto a mass spectrometer disposed in a vacuum chamber where the massspectrum is given. This electrospray ionization mass analysis apparatusis characterized in that the ion current value of a specific mass ismonitored more than once for each sample, and an approximate expressionis formed from multiple ion current values monitored subsequent tomeasurement of multiple samples, to predict the number of samplemeasurements where the ion current value is below the threshold value,whereby a warning is displayed on a CRT.

The ESI operates as follows: Voltage of several kilovolts is appliedbetween a metallic capillary having an inner diameter of about 0.1 mmand a counter electrode arranged at some distance (about several tens ofmm) away therefrom. When a sample solution is led to the metalliccapillary and a high voltage is applied, the liquid in the capillary isdielectrically polarized at the capillary outlet by a high electricfield formed on the tip of a metallic capillary. In the positiveionization mode, positive electric charge is induced on the liquidsurface, while in the negative ionization mode, negative electric chargeis induced on the liquid surface.

As a result, a conical liquid called Taylor cone is pulled out into theatmosphere from the capillary outlet by electric field. If electricfield is stronger than the surface tension at the tip of the Taylorcone, electrically charged extremely fine droplets are released into theatmosphere from the tip of the Taylor cone. In conformity to electricfield, the generated charged droplets fly in the atmosphere toward acounter electrode to repeat collision with molecules in the atmosphere.This allows charged droplets to be mechanically broken, and evaporationof solvent from the droplet surface is promoted so that charged dropletsare quickly pulverized. In the final stage, ions in charged droplets arereleased into the atmosphere. The ion flies in the atmosphere toward acounter electrode and is led into a highly vacuum mass spectrometerthrough a capillary tube or aperture arranged in the counter electrodewhere it is subjected to mass analysis.

Further, the present invention provides an electrospray ionization massanalysis apparatus wherein eluate from a chromatograph is introducedinto the capillary tube, and an electrospray ion source arranged forgenerating ions under atmospheric pressure generates ions, which are ledinto a mass spectrometer disposed in a vacuum chamber where the massspectrum is given. This electrospray ionization mass analysis apparatusis characterized by sequentially comprising:

a step of introducing the aforementioned sample into the injector andmicro column of the chromatograph in that order,

a step of separating the sample for each component and ionizing it afterfeeding into the aforementioned ion source in conformity to the lapse oftime,

a step of repeating mass sweeping with the aforementioned massspectrometer and storing the collected mass spectra into the controldata processor,

a step of measuring the current value (Is) of the ion having a specificmass in the sample, and comparing between the measured Is and thethreshold value (It),

a step of continuing measurement if Is exceeds It,

a step of completing measurement if the Is is not below the It by thetime the aforementioned measurement terminates, and starting measurementof the next sample,

a step of indicating an error through the control data processor if theerror has occurred where the Is is reduced below the It due to suddenreduction of the Is, and specifying the action to be taken to correctthe error,

a step of giving a command of suspending start of sweeping to the masssweep power source of the mass spectrometer to suspend the collection ofmass spectra,

a step of recording an error in the data and displaying that warning,and

a step of suspending transmission of the signal for starting the nextsample measurement to an automatic sampler.

Further, the present invention provides an electrospray ionization massanalysis apparatus similar to the above characterized by sequentiallycomprising:

a step of introducing the aforementioned sample into the injector andmicro column of the chromatograph in that order,

a step of separating the sample for each component and ionizing it afterfeeding into the aforementioned ion source in conformity to the lapse oftime,

a step of repeating mass sweeping with the aforementioned massspectrometer and storing the collected mass spectra into the controldata processor,

a step of measuring the current value (Is) of the ion having a specificmass, and comparing between the measured Is and the threshold value(It),

a step of continuing measurement if Is exceeds It,

a step of completing measurement if the Is is not below the It by thetime the aforementioned measurement terminates, and starting measurementof the next sample,

a step of suspending the collection of mass spectra due to abruptreduction of the Is,

a step of indicating an error without suspending the collection of massspectra during the measurement of one sample by liquid chromatograph(LS),

a step recording an error of the Is having reduced below the It, andterminating the data file upon completion of the LC measurement,

a step of instructing suspension of starting the measurement of the nextsample if an error is displayed, and

a step of instructing the automatic sampler to start the measurement ofthe next sample if no error is indicated.

Further, the present invention provides an electrospray ionization massanalysis apparatus similar to the above characterized by sequentiallycomprising:

a step of introducing the aforementioned sample into the injector andmicro column of the chromatograph in that order,

a step of separating the sample for each component and ionizing it afterfeeding into the aforementioned ion source in conformity to the lapse oftime,

a step of repeating mass sweeping with the aforementioned massspectrometer and storing the collected mass spectra into the controldata processor,

a step of measuring the current value (Is) of the ion having a specificmass, and comparing between the measured Is and the threshold value(It),

a step of continuing measurement if Is exceeds It,

a step of completing measurement if the Is is not below the It by thetime the aforementioned measurement terminates, and starting measurementof the next sample,

a step of measuring the Is at least once for each supply of the sampleimmediately before the column is brought into equilibrium by the solventof the mobile phase prior to supply of the sample,

a step of recording and displaying an error when the Is is reduced belowthe It,

a step of stopping the measurement and suspending the supply of a newsample, and

a step of continuing the measurement if the Is is above the It.

As described above, the present invention monitors the Na⁺ ion thatsurely occurs in the ESI. When it has been reduced below the thresholdvalue, measurement is stopped because clogging is assumed to haveoccurred. Further, the current value of the Na⁺ ion is collected foreach measurement and the time for reduction below the threshold value isestimated from their changes. This is indicated on the CRT or the like.In other words, in the micro LC and ESI, clogging of the ESI nozzle andcapillary tube seriously deteriorates the throughput and datareliability. Sample waste can be minimized and reliability of acquireddata can be improved by detecting this clogging and stopping measurementand introduction of the sample. Maintainability can be improved bypredicting possible clogging.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an overall configuration drawing of electrospray ionizationmass analysis apparatus as an embodiment of the present invention;

FIG. 2 is a configuration drawing representing a micro LC and ESI ionsource as embodiments of the present invention;

FIG. 3 is a drawing representing the operation flow as an embodiment ofthe present invention;

FIG. 4 is another drawing representing the operation flow as anembodiment of the present invention;

FIG. 5 is a further drawing representing the operation flow as anembodiment of the present invention;

FIG. 6 is an explanatory diagram of a mass spectrum in the ESI positiveion mode;

FIG. 7 is an explanatory diagram of a mass spectrum in the ESI negativeion mode;

FIG. 8 is an explanatory diagram representing the measurement operationaccording to the present invention;

FIG. 9 is an explanatory diagram of a mass chromatogram according to thepresent invention;

FIG. 10 is an explanatory diagram of a mass chromatogram according tothe present invention when the ESI nozzle is clogged in the middle ofmeasurement;

FIG. 11 is an explanatory diagram of a mass chromatogram according tothe present invention when the ESI nozzle is clogged from the start ofmeasurement;

FIG. 12 is an explanatory diagram representing the method for predictingthe clogging of the ESI nozzle;

FIG. 13 is an explanatory diagram representing the measurement operationusing ions trap mass spectrometer according to the present invention;and

FIG. 14 is another explanatory diagram representing the measurementoperation using ions trap mass spectrometer according to the presentinvention.

BEST FORM OF EMBODIMENT OF THE PRESENT INVENTION

FIG. 1 is an overall configuration drawing of electrospray ionizationmass analysis apparatus as an embodiments of the present invention.Solution containing sample component separated by the micro LC 1 is sentto the ESI probe 3 of the ESI ion source 4 through a capillary tube 2.The ESI probe 3 is arranged on the XYZ three-axis positioning device 9.The sample solution sent from the micro LC 1 is fed to the ESI capillarytube nozzle 48 constituting the ESI probe 4 and a spray ion flow isformed as charged droplet 6 sprayed into the atmosphere from the nozzletip. The charged droplet 6 is discharged in the form of ions into theatmosphere and is fed into a vacuum chamber 12 through a capillary tube8 arranged on a vacuum partition 11. The mass analysis apparatus iscomposed of vacuum chambers 12, 15 and 19 having different pressures,and each chamber is evacuated by each of independent vacuum pumps 20, 21and 22.

A skimmer is provided in the vacuum chamber 12, and ions guide 16 isarranged in the vacuum chamber 15. A mass spectrometer 17 and a detector18 are disposed in the vacuum chamber 19 kept high vaccum. Ions are ledinto a mass spectrometer 17 through ions guide 16, and are subjected tomass analysis. When the voltage supplied from the mass sweep powersupply 23 is swept, the ions are separated according to each mass, andion current is detected by a detector 18. Ion current signalcorresponding to each mass is fed to a control data processor 24, whereit is collected as a mass spectrum.

The ion guide 16 consists of cylindrical electrodes formed by four, sixand eight metallic rods arranged on a certain circumference at anequally spaced interval. These rods are wired alternately and highfrequency is applied between two electrodes. When the ion is led ontothe center axis of this ion guide, the ion is subjected to vibration byhigh frequency and is brought into collision with gas molecule to beconverged on the ion guide axis. Ion can be transferred by this ionguide without being lost.

The capillary tube 8 is a pipe made of stainless steel, other metal orglass. Preferably, it has an inner diameter of 0.4 to 0.3 mm and alength of 10 cm. It is used with a heater disposed around it forheating.

In the ion trap mass spectrometer, the mass spectrometer 17 is composedof three electrodes as rotary symmetric elements of hyperbolic form anda toroidal ring electrode and two end cap electrodes sandwiching a ringelectrode regionrranged. When main high frequency voltage is applied tothe ring electrode from the main high frequency power source 23, aquadrupole electric field is formed in the space formed by theaforementioned three electrodes. The ion generated by the ESI ion sourceis fed to the vacuum space to reach the ion trap mass spectrometerthrough the skimmer and ion guide. Ions gate electrode is arranged infront of the ion trap electrode so that ion is led in ions trap or isblocked therefrom.

When voltage with the same polarity as that of the ion is applied to theion gate electrode, ion will be blocked, namely, the ion gate is turnedoff. Conversely when voltage with the polarity reverse to that of theion is applied, ion is led into the ion trap.

Ion can be also be stored in the ion trap as it is introduced for apredetermined time when the main high frequency wave is applied to thering electrode. This ensures the average mass spectrum to be formed evenif the amount of ion in the ion source fluctuates. The mass spectrum canbe formed by performing MS/MS with the ion gate turned off and sweepingthe main high frequency voltage applied to the ring electrode.

In the figure, numeral 4 denotes a ESI ion source, 5 a high voltagepower source, 6 a spray ion flow, 7 a ion source space, 13 a skimmer, 14a vacuum partition, 18 a detector, 49 a waste water bottle.

FIG. 2 is a configuration drawing representing a micro LC 1 and ESI ionsource. Two solvents in mobile phase are stored in solvent bottles 40and 41, respectively. A micro mixer 44 mixes two solvents sucked anddelivered by two pumps 42 and 43. The percentage composition of twosolvents can be controlled by changing the amount delivered from thepump in conformity to the gradient parameter. The mixed solvents ofmobile phase are split by a next flow splitter. The split ratio isnormally set to 1/10 through 1/100, and can be set from the outside. Thesolvent of mobile phase split to 1 μL/min. through 10 μL/min. is fed tothe micro column 47 through an injector 46.

Sample solution is introduced by the injector, and is separated by amicro column 47 for each component. The separated component is fed tothe ESI nozzle 48. A high voltage of about several kV is supplied to theESI nozzle 48 from a high voltage power supply 5. The sample solution issprayed and ionized into the atmosphere by the high electric fieldgenerated on the tip of the ESI nozzle 48. The positive/negativeionization mode can be switched by changing over the polarity of highvoltage applied to ESI nozzle 48.

FIG. 3 is a flow chart representing the operation according to repeatedmass spectrum collection and Na⁺ ion monitoring method. The LC/MSanalysis starts and the sample is fed to a micro column 47 from theinjector 46. The sample is separated for each component, and is fed tothe ESI ion source 4 with the lapse of time, where it is ionized. Themass spectrometer repeats mass sweeping, and the mass spectra arecollected repeatedly. The mass spectrum is stored in the control dataprocessor.

The current value I₂₃ of the ion of mass 23 is compared with thethreshold value It. If I₂₃ has exceeded It (I₂₃>It), measurement iscontinued. If the I₂₃ is not reduced below the It by the time themeasurement terminates, measurement is assumed to have been completedcorrectly. The data are processed and the file is terminated. This isfollowed by the step of measuring the next sample.

If the micro column is clogged at a certain time period, the amount ofNa⁺ ion undergoes an abrupt reduction, with the result that measurementI₂₃ is reduced below It (I₂₃<It). The control data processor sets anerror flag to start taking action against the error. Namely, action istaken to ensure that sweep start command is not sent to a mass sweeppower source 23, whereby collection of mass spectrum is suspended.Further, abnormal suspension is recorded in the data and the file isterminated. An error message is displayed on the CRT.

Even after completion of the LC measurement of this sample, the controldata processor does not sent to the automatic sampler the signal tostart the measurement of the next sample. This suspends introduction ofthe sample after the error has occurred, with the result that waste ofthe sample can be avoided.

FIG. 4 is a flow chart representing the operation according to themethod where an error state is recorded in the data without suspendingthe measurement despite occurrence of the error status. In the exampleshown in FIG. 3, the collection of the mass spectrum was suspended whenan abnormal reduction of I₂₃ due to clogging of the micro column hadbeen detected. In FIG. 4, however, the collection of mass spectrum isnot suspended as long as the LC measurement of one sample continues, andan error flag is set. When the LC measurement has terminated at theexpiration of the measurement time, a error status is recorded in thedata to indicate that the

I₂₃ has reduced below the It. Then the data file is terminated. Here theerror flag is checked. If an error flag is set, the measurement of thenext sample is not started. If no flag is set, it sends to the automaticsampler a command to start the measurement of the next sample.

FIG. 5 is a flow chart representing the operation according to themethod where the I₂₃ is monitored before the sample is supplied. Theclogging of the micro column, pipe or ESI nozzle may be monitored onceor several times for each sample supply by the I₂₃, without beingmonitored at all times. Generally, in order to ensure measurementcharacterized by excellent reproducibility in the LC analysis,initialization of LC is essential; namely, the column must beequilibrated by the solvent of mobile phase before the sample issupplied. It is also possible to monitor Na⁺ and Cl⁻ ions immediatelybefore this initialization terminates.

When a series of samples are to be measured, initialization is oftencarried out under one and the same conditions. Then ion monitoringconditions are matched conveniently. If the I₂₃ is reduced below thethreshold value due to clogging, an error flag is set. The error isrecorded in the data and the error status is displayed on the CRT. Thenthe measurement is stopped. In other words, no new sample is supplied.If the I₂₃ is greater than the threshold value, measurement iscontinued. Judgment of I₂₃ against noise can be reinforced by taking anaverage of ion current values of I₂₃ instead of one. Further, the ioncan be monitored once at predetermined time intervals, e.g. every tenminutes.

FIG. 6 is an explanatory diagram of a typical mass spectrum in the ESIpositive ion mode. Generally, the mass spectrum is composed of many ionspecies. In the low mass region, ammonium ion NH4 with a mass number ofseveral m/z=18, alkali metal ion such as Na⁺ ion with a mass number ofm/z=23, or (NH₄ ⁺+S)⁺ and (Na⁺S)⁺ ions formed by adding solventmolecular S to these ions often appear. The sample led to the ESI ionsource is a mixture in many cases. In such cases, ions (I+H)⁺ derivedfrom impurities in sample appears. Further, pseudomolecular ion (M+H)⁺derived from the main component and fragment ion (M+H−N)⁺ formed bycleavage of pseudomolecular ion appear. (M+H+S)⁺ and the like formed byaddition of solvent molecule to the pseudomolecular ion appear in thehigher mass the mass of region than pseudomolecular ion.

FIG. 7 is an explanatory diagram of a mass spectrum in the ESI negativeion mode. Similarly to the case of positive ion mode, the mass spectrumconsists of many ion species. Cl⁻, SO₄H⁻, (S−H)⁻ and ions formed byadding solvent molecules to these ions appear in the low mass region,Further, ions (I−H)⁻ derived from the impurities in sample appear.Pseudomolecular ion (M−H)⁺ derived from the main component and fragmention (M−H+N)⁻ formed by cleavage of pseudomolecular ion appear. (M−H+S)⁻and the like formed by addition of solvent molecule to thepseudomolecular ion appear in the higher-mass region than the mass ofpseudomolecular ion.

As described above, in the low mass region, Na⁺ and Cl⁻ ions unrelatedto the sample or mobile phase are often observed. This is because atrace quantity of NaCl and other salts as impurities are present in thesample and solvent, and LC/MS apparatus is slightly contaminated byNaCl, etc. Especially the Na⁺ and Cl⁻ ions are not generated in theatmospheric chemical ionization (APCI) resulting from corona discharge;Na⁺ or Cl⁻ ions pertain to ion species which can be detected only byESI. Consequently, when the apparatus is started, a person in charge ofmeasurement can make sure of the smooth operation of the apparatus byintroducing only the solvent into the ESI ion source and observing thepresence of Na⁺ or Cl⁻ ions in the mass spectrum.

In the present invention, Na⁺ ion is observed in the positive ion modeand Cl⁻ ion is observed in the negative ion mode. By checking if the ioncurrent value exceeds the threshold value, evaluation is made todetermine if the ESI is correctly operating or not.

In the positive ion mode, NH₄ ⁺ or the like can be adopted as ionspecies to be monitored. It is possible to mix a trace quantity oftriethylamine in the mobile phase and to monitor its pseudomolecular ion(C₂H₅)₃NH⁺. Namely, for the ion species to be monitored, the mass can beselected and set in response to measurement.

FIG. 8 is a schematic diagram representing the measurement operation.Mass sweeping is repeated at predetermined intervals 0 to t₁, t₁ to t₂,and t₂ to t₃. According to this mass sweeping, the mass spectrum iscollected repeatedly. Assume that ion a is the ion to be monitored. Itis observed on the mass spectrum, independently of the presence orabsence of the sample component. This ion current is traced to create amass chromatogram. When the sample component is introduced into the ESIion source, pseudomolecular ion b is increased by the correspondingamount.

FIG. 9 is a diagram showing the result arranged in the form of a masschromatogram by the control data processor. The chromatogram on theupper stage of FIG. 9 is formed by tracing the integration of the ioncurrent in a certain range. It is called total ion chromatogram (TIC).Here three components are detected. Although there is a slight wavinessor fluctuation of Na⁺ ion while three components are eluted, an almostflat mass chromatogram is provided. This shows that the micro LC and ESIare operating properly.

FIG. 10 shows an example when the ESI nozzle is clogged in the middle ofmeasurement. It is estimated that Na⁺ ion current is reduced to zero inthe middle of measurement, with the result that the ESI nozzle isclogged. In TIC, on the other hand, the components eluted beforeclogging are detected as a peaks. If clogging occurs in the middle ofmeasurement, the sample component is not introduced into the ESI ionsource, so the subsequent components are not detected. The base line istraced by the ITC. If only the TIC trace is observed without the Na⁺ ionbeing monitored, it is highly possible to arrive at a misunderstandingthat this sample originally contains only one component. An error can beeasily identified by measurement of the Na⁺ ion. Consequently, thesupply and measurement of the next sample are stopped, whereby waste ofa precious sample can be avoided. If the measurement is continuedwithout Na⁺ ion being monitored, a large volume of meaningless datacorresponding to that of FIG. 11 will be stored in the control dataprocessor. Not only that, the sample will be wasted. In this case, theperson in charge of measurement will find it difficult to determine ifsuch data has been formed because the sample had not originally includedthe components to be measured, or measurement has not been carried outappropriately.

FIG. 12 is another embodiment of the present invention. Clogging of themicro column or ESI may occur suddenly, but in many cases, non-volatilecomponents are deposited on the inner wall of the capillary tubegradually to clog the capillary tube in the final stage. If gradualnarrowing of the capillary tube can be predicted in advance, the personin charge of measurement can feel easy about proceeding withmeasurement.

FIG. 12 is a diagram representing the relationship between the ioncurrent of Na⁺ ion and number of measurements. In the step ofinitialization prior to supply of the sample, the current of a specificion is monitored and is recorded by the control data processor. Thecorrelation between the ion current value and the number of measurements(n) is found out. If the slope of this primary function formed from thecorrelation is negative, an approximate function is extrapolated, and acrossing point “n” with the level of clogging (TL) is formed. Thus, thedifference (n−P) from the current measuring point P, namely, thepredicted point “n” denotes the number of times before clogging occurs.If (n−P) has a sufficient margin, measurement can be continued. However,if (n−P) is reduced, an alarm is issued to the CRT or the like, and themeasurement of precious samples can be avoided in this stage. It is alsopossible to prepare or replace the column, capillary tube or nozzle atan earlier stage.

FIGS. 13 and 14 show a further embodiment of the present invention. Massspectrometers that are based on a different principle as a LC/MS is usedat present. They include a quadrupole MS (QMS), magnetic field type MS,TOF, ion trap MS, and ion cyclotron resonance MS (ICRMS). The ion trapMS and ion cyclotron resonance MS (ICRMS) regionlso called ion storagetype MS, based on the operating principle different from those of otherMSs.

The ion trap MS is a small sized MS where two end cap electrodes ofrotary hyperbolic surface are opposed to each other so as to sandwichthe toroidal ring electrode. The main high frequency voltage is appliedto the ring electrode and ions are trapped in ions trap space enclosedby three electrodes. Then the main high frequency voltage is swept, andions are released from the ion trap space sequentially in the order ofmass. The mass spectrum can be formed by detecting the released ion.Unlike the QMS or the like, the ion trap MS allows ionintroduction/storage and mass sweep/mass spectrum acquisition to beperformed on a time division basis. As shown in FIG. 13,

“0 to t₁” is the time period for ion introduction and storage, when theion generated by the ESI ion source is introduced and stored into theion trap space. During this time, the main high frequency voltage is setat a lower level so that ions over a wide mass range can be trapped.During the time period of “t₁ to t₂”, ion introduction is stopped, andthe main high frequency voltage is swept to acquire the mass spectrum.

Namely, during the period of 0 to t₂, one mass spectrum is acquired.This step is repeated to perform LC/MS measurement. Na⁺ ion has a massof 23. The main high frequency voltage (referred to as “IL”) to be setduring the ion introduction and storage period must be a low voltagewhere Na⁺ ion can be trapped. The maximum mass that allows an effectivetrapping of ions into the ion trap is assumed as about 30 times the IL.If the IL is 20 to ensure that Na⁺ ion can be trapped, the maximum masswill be 20*30=600. If the IL is reduced to ensure that Na⁺ ion can betrapped, then ions of peptide and protein having a mass of 600 orgreater cannot be trapped; namely, they cannot be measured. Symbol “a”in FIG. 13 denotes the Na⁺ ion. The ion “b” having a mass of 600 orsmaller can be measured.

FIG. 14 shows a method for measuring a high mass ion and Na⁺ ion. The “0to t₁” indicates the time period for ion introduction and storage.During this period,

The ion level IL1 is set to 20 or smaller, and Na⁺ ion is trapped.During the period “t₁ to t₂”, the main high frequency voltage is sweptand the current value I₂₃ of Na⁺ ion is formed. Then during the period“t₂ to t₃”, the ion level (IL2) is set to about 70 to provide against ahigh mass sample. This will allow ions having a mass of 70 to about 2000to be trapped. During the period “t₃ to t₄”, the mass from 70 to 2,000is swept to get the mass spectrum. In the period “t₄ to t_(n)”,successive trapping of high mass ions and acquisition of mass spectrumare repeated. The cycle of mass spectrum acquisition is about 0.2seconds, so clogging can be detected even if Na⁺ ion is assumed to bemonitored once for 100 acquisitions of high mass spectrum.

In the case of ion trap MS, monitoring of Na⁺ ion of low mass andacquisition of high mass spectrum can be made compatible by adjustingthe ion level IL.

Comparison between ion current value and threshold value is intended todistinguish between the noise of the detector and actual signals. Thesetting of the threshold value can be changed in conformity to theconditions of the apparatus.

The above has mainly described the coupling between the ESI and microLC. The present invention is also applicable to the coupling betweenvarious types of chromatography including the conventional LC,semi-micro LC, micro LC and CE, and ESI or its improved ionization artsincluding ion spray, sonic spray and nano spray.

It has been described in the above that reduction of the I₂₃ is mainlycaused by clogging of the capillary tube. Reduction of the I₂₃ can alsobe caused by fluctuation of spraying due to contamination of the ESInozzle tip or deflection of the spray direction. In this case, there isa substantial reduction in the ion current value of the component to bemeasured. Consequently, it is still an effective method to monitor theI₂₃ and, if the result is below the threshold value, it is assumed as anerror, even if it results from different causes.

In the above description, Na⁺ ion is used to explain the ion species tobe monitored. Cl³¹ or other ion species (e.g. NH₄ ⁺) may also be used.Any ion species will be acceptable if it is stably present duringmeasurement, independently of LC conditions. So in addition to the Na⁺occurring as background ion, it is also possible to use the NH₄ ⁺ ionthat appears by mixing a very small amount of ammonium acetate CH₃CO₂NH₄or the like in the LC eluent.

Industrial Field of Application

Since the flow rate of the micro LC/MS is low, the dead volume must beminimized or the diameter of the capillary tube must be reduced.Further, the sample and salt are likely to deposit in the capillary tubedue to low flow rate, with the result that clogging of the capillarytube and ESI nozzle often occurs. The present invention provides anelectrospray ionization mass analysis apparatus and its system thatensures clogging to be predicted, or detected immediately when it hasoccurred, thereby suspending measurement to prevent samples from beingwasted, and improving the reliability of the formed data and themaintainability through earlier replacement of a clogged component.

What is claimed is:
 1. An electrospray ionization mass analysisapparatus wherein a sample solution from a chromatograph is introducedinto a capillary tube, and an electrospray ion source arranged forgenerating ions under atmospheric pressure generates ions, which are ledinto a mass spectrometer disposed in a vacuum chamber where said ionsare subjected to mass analysis; said electrospray ionization massanalysis apparatus further characterized in that; the current value orstrength of the ion having a specified mass in said sample solution ismeasured, and, when said current value has reduced below a thresholdvalue, an error state is displayed.
 2. An electrospray ionization massanalysis apparatus according to claim 1 characterized in that saidcurrent value or strength are measured prior to supply of said sample,and said current value is compared with a threshold value.
 3. Anelectrospray ionization mass analysis apparatus according to claim 2characterized in that comparison between said ion current value withsaid threshold value is carried out at a predetermined intervalsubsequent to supply of said sample.
 4. An electrospray ionization massanalysis apparatus according to claim 3 characterized in that said errorstatus is recorded in the data.
 5. An electrospray ionization massanalysis apparatus according to claim 4 characterized in that, when saiderror status is displayed, the data is saved and the measurement in massanalysis is then suspended.
 6. An electrospray ionization mass analysisapparatus according to claim 5 characterized in that, when said errorstatus is displayed prior to supply of said sample, a command is issuedto suspend supply of said sample.
 7. An electrospray ionization massanalysis apparatus according to claim 6 characterized in that thesetting of the mass of said ion for monitoring said measured ion currentvalue or strength can be changed from the outside.
 8. An electrosprayionization mass analysis apparatus according to claim 7 characterized inthat the setting of said threshold value can be changed from theoutside.
 9. An electrospray ionization mass analysis apparatus accordingto claim 8 characterized in that, in the positive ion measurement mode,the mass of said ion to be monitored is
 23. 10. An electrosprayionization mass analysis apparatus according to claim 9 characterized inthat, in the negative ion measurement mode, the mass of said ion to bemonitored is
 35. 11. An electrospray ionization mass analysis apparatuswherein a sample solution from a chromatograph is introduced into acapillary tube, and an electrospray ion source arranged for generatingions under atmospheric pressure generates ions, which are led into amass spectrometer disposed in a vacuum chamber where said ions aresubjected to mass analysis; said electrospray ionization mass analysisapparatus further characterized in that; the current value or strengthof the ion having a specified mass in said sample solution is measuredand stored for multiple samples, the number of measurements where saidion current value or strength is below said threshold value is predictedbased on the relationship between said multiple ion current values orstrengths and the number of measurements, and an error state isdisplayed in conformity to said predicted number of measurements.
 12. Anelectrospray ionization mass analysis apparatus according to claim 11characterized in that said ion current value is measured prior to supplyof said sample.
 13. An electrospray ionization mass analysis apparatuswherein; a sample solution from a chromatograph is introduced into acapillary tube, and an electrospray ion source arranged for generatingions under atmospheric pressure generates ions, which are led into amass spectrometer disposed in a vacuum chamber where said ions aresubjected to mass analysis; said electrospray ionization mass analysisapparatus comprising ion level setting means where, during the time whenthe current value of the ion having a specific mass is measured, thelevel of the ion to be trapped is set to a level lower than that of saidspecific mass and, at other times, the level of the ion to be trapped isset to a level higher than that of said specific mass.
 14. Anelectrospray ionization mass analysis apparatus wherein a samplesolution separated from a micro liquid chromatograph arranged forseparating a sample solution is introduced into a capillary tube, highvoltage is applied from a high voltage power source connected to the tipof said capillary tube and a counter electrode having an aperture;whereby a spray ion flow is generated from the tip of said capillarytube toward said aperture by an electrospray ion source, said ion flowgenerated by said ion source is introduced through said aperture to askimmer cone and ion guide disposed in a vacuum chamber, and then toions storage type mass spectrometer, where said ion is subjected to masssweeping and is detected by a detector to obtain mass spectrum; saidelectrospray ionization mass analysis apparatus further characterized inthat the current value or strength of the ion having a specified mass insaid sample solution is measured, and, when said current value hasreduced below a threshold value, an error state is displayed.
 15. Anelectrospray ionization mass analysis apparatus according to claim 14characterized in that said skimmer, ion guide and ion storage type massspectrometer are each disposed integrally in each vacuum chamber, whichis provided with a vacuum pump.
 16. An electrospray ionization massanalysis apparatus according to claim 15 characterized by comprising anXYZ3 axis positioner for setting said spray ion flow with respect tosaid capillary tube.
 17. An electrospray ionization mass analysisapparatus according to claim 13 or 14 characterized in that said ionstorage type mass spectrometer is ions trap mass spectrometer.
 18. Anelectrospray ionization mass analysis apparatus according to claim 13 or14 characterized in that said ion storage type mass spectrometer is ionscyclotron resonance (ICR) mass spectrometer.
 19. An electrosprayionization mass analysis system wherein; a sample solution from achromatograph is introduced into a capillary tube, and high voltage isapplied to the tip of said capillary tube under atmospheric pressurewhereby spray ions are generated, and are then led into a massspectrometer disposed in a vacuum chamber where said ions are subjectedto mass analysis; said electrospray ionization mass analysis apparatusfurther characterized in that; the current value or strength of the ionhaving a specified mass in said sample solution is measured, and, whensaid current value has reduced below a threshold value, an error stateis displayed.
 20. An electrospray ionization mass analysis systemwherein; a sample solution from a chromatograph is introduced into acapillary tube, and an electrospray ion source arranged for generating aspray ion wider atmospheric pressure generates ions, which are led intoa mass spectrometer disposed in a vacuum chamber where said ions aresubjected to mass analysis; said electrospray ionization mass analysisapparatus further characterized in that; the current value or strengthof the ion having a specified mass in said sample solution is measuredand stored for multiple samples, the number of measurements where saidion current value or strength is below said threshold value is predictedbased on the relationship between said multiple ion current values orstrengths and the number of measurements, and an error state isdisplayed in conformity to said predicted number of measurements.
 21. Anelectrospray ionization mass analysis system wherein; a sample solutionfrom a chromatograph is introduced into a capillary tube, and anelectrospray ion source arranged for generating ions under atmosphericpressure generates ions,, which are led into an ion trap massspectrometer disposed in a vacuum chamber where said ions are subjectedto mass analysis; said electrospray ionization mass analysis apparatusfurther characterized in that; during the time when the current value orstrength of the ion having a specific mass in said spray ion aremeasured, the level of the ion to be trapped is set to a level lowerthan that of said specific mass and, when they are not measured, thelevel of the ion to be trapped is set to a level higher than that ofsaid specific mass.
 22. An electrospray ionization mass analysisapparatus wherein; a sample solution from a chromatograph is introducedinto a capillary tube, and an electrospray ion source arranged forgenerating ions under atmospheric pressure generates ions, which are ledinto a mass spectrometer disposed in a vacuum chamber where said ionsare subjected to mass analysis; said electrospray ionization massanalysis apparatus further characterized by sequentially comprising: astep of introducing said sample into the injector and micro column ofthe chromatograph in that order, a step of separating the sample foreach component and ionizing it after feeding into said ion source inconformity to the lapse of time, a step of repeating mass sweeping withsaid mass spectrometer and storing the collected mass spectra into thecontrol data processor, a step of measuring the current value (Is) ofthe ion having a specific mass in the sample, and comparing between themeasured Is and the threshold value (It), a step of continuingmeasurement if Is exceeds It, a step of completing measurement if the Isis not below the It by the time said measurement terminates, andstarting measurement of the next sample, a step of indicating an errorthrough the control data processor if the error has occurred where theIs is reduced below the It due to sudden reduction of the Is, andspecifying the action to be taken to countermeasure against the error, astep of giving a command of suspending start of sweeping to the masssweep power source of the mass spectrometer to suspend the collection ofmass spectra, a step of recording an error in the data and displayingthat warning, and a step of suspending transmission of the signal forstarting the next sample measurement to an automatic sampler.
 23. Anelectrospray ionization mass analysis system wherein; a sample solutionfrom a chromatograph is introduced into a capillary tube, and anelectrospray ion source arranged for generating ions under atmosphericpressure generates ions, which are led into a mass spectrometer disposedin a vacuum chamber where mass spectrum is given; said electrosprayionization mass analysis apparatus further characterized by sequentiallycomprising: a step of introducing said sample into the injector andmicro column of the chromatograph in that order, a step of separatingthe sample for each component and ionizing it after feeding into saidion source in conformity to the lapse of time, a step of repeating masssweeping with said mass spectrometer and storing the collected massspectra into the control data processor, a step of measuring the currentvalue (Is) of the ion containing a specific mass, and comparing betweenthe measured Is and the threshold value (It), a step of havingmeasurement if Is exceeds It, a step of completing measurement if the Isis not below the by the time said measurement terminates, and startingmeasurement of the next sample, a step of suspending the collection ofmass spectra due to abrupt reduction of the Is, a step of indicating anerror without suspending the collection of mass spectra during themeasurement of one sample by liquid chromatograph (LC), a step ofrecording an error of the Is having reduced below the It, andterminating the data file upon completion of the LC measurement, a stepof instructing suspension of starting the measurement of the next sampleif an error is displayed, and a step of instructing the automaticsampler to start the measurement of the next sample if no error isindicated.
 24. An electrospray ionization mass analysis system wherein;a sample solution from a chromatograph is introduced into a capillarytube, and an electrospray ion source arranged for generating ions underatmospheric pressure generates ions, which are led into a massspectrometer disposed in a vacuum chamber where mass spectrum is given;said electrospray ionization mass analysis apparatus furthercharacterized by sequentially comprising: a step of introducing saidsample into the injector and micro column of the chromatograph in thatorder, a step of separating the sample for each component and ionizingit after feeding into said ion source in conformity to the lapse oftime, a step of repeating mass sweeping with said mass spectrometer andstoring the collected mass spectra into the control data processor, astep of measuring the current value (Is) of the ion having a specificmass, and comparing between the measured Is and the threshold value(It), a step of continuing measurement if Is exceeds It, a step ofcompleting measurement if the Is is not below the It by the time saidmeasurement terminates, and starting measurement of the next sample, astep of measuring the Is at least once for each supply of the sampleimmediately before the column is brought into equilibrium by the solventof the mobile phase prior to supply of the sample, a step of recordingand displaying an error when the Is is reduced below the It, a step ofstopping the measurement and suspending the supply of a new sample, anda step of continuing the measurement if the Is is above the It.